1. Field of the Invention
The present invention relates to the field of molecular biology and nucleic acid chemistry. More specifically, it relates to methods and reagents for amplifying Neisseria gonorrhoeae nucleic acid. The invention therefore has applications in the detection of Neisseria gonorrhoeae, the field of medical diagnostics generally, and the field of molecular biology.
2. Description of Related Art
Neisseria gonorrhoeae is the causitive agent of gonorrhea, one of the most commonly reported bacterial infections in the United States.
Miyada and Born, 1991, Mol. Cell. Probes 5:327-335; U.S. Pat. No. 5,256,5365; and U.S. Pat. No. 5,525,717; each incorporated herein by reference, describe the detection of N. gonorrhoeae using a DNA probe derived from a genomic fragment containing an open reading frame (ORF 1) having significant homology with the sequence of the N. gonorrhoeae cytosine DNA methyltransferase gene (M.Ngo PII). Probes derived from this sequence were shown to hybridize to purified DNA from 105/106 N. gonorrhoeae strains tested in a dot blot format. Cross-reactivity with other Neisseria species was observed only with N. mucosa, however this cross-reactivity was eliminated using a high stringency wash.
The invention of the methods for amplifying specific sequences of nucleic acids, in particular, the polymerase chain reaction (PCR), makes possible the rapid detection of nucleic acids present in a sample in what was previously an undetectably low quantity (see U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188, each of which is incorporated herein by reference). The development and application of PCR are described extensively in the literature. For example, a range of PCR-related topics are discussed in PCR Technology--principles and applications for DNA amplification, 1989, (ed. H. A. Erlich) Stockton Press, New York; PCR Protocols: A guide to methods and applications, 1990, (ed. M. A. Innis et al.) Academic Press, San Diego; and PCR Strategies, 1995, (ed. M. A. Innis et al.) Academic Press, San Diego; each of which is incorporated herein by reference. Commercial vendors, such as Perkin Elmer (Norwalk, Conn.), market PCR reagents and publish PCR protocols.
Since the original description of nucleic acid amplification, various primer-based nucleic acid amplification methods have been described including, but are not limited to, Ligase Chain Reaction (LCR, Wu and Wallace, 1989, Genomics 4:560-569 and Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193); Polymerase Ligase Chain Reaction (Barany, 1991, PCR Methods and Appl. 1:5-16); Gap-LCR (PCT Patent Publication No. WO 90/01069); Repair Chain Reaction (European Patent Publication No. 439,182 A2), 3SR (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177; Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878; PCT Patent Publication No. WO 92/0880A), and NASBA (U.S. Pat. No. 5,130,238). All of the above references are incorporated herein by reference. A survey of amplification systems is provided in Abramson and Myers, 1993, Current Opinion in Biotechnology 4:41-47, incorporated herein by reference.
Purohit and Silver, U.S. Pat. No. 5,550,040, incorporated herein by reference, describe the detection of N. gonorrhoeae using a PCR amplification/detection assay. Based on a comparison of DNA sequences from various different strains of N. gonorrhoeae described in the literature, primers were designed to amplify a 201 nucleotide target sequence within the sequence provided by Miyada and Born, supra. The primers amplified the target sequence from all strains of N. gonorrhoeae tested, although DNA from some strains of N. mucosa was also amplified. The amplified product was then hybridized with an N. gonorrhoeae-specific probe to achieve the desired specificity. The resulting amplification/detection assay was observed to detect all strains of N. gonorrhoeae tested without cross-reactivity with non-N. gonorrhoeae DNA.
Patients infected with N. gonorrhoeae often also are infected with Chlamydia trachomatis. The N. gonorrhoeae primers described in the '040 patent were used in conjunction with primers for the amplification of C. trachomatis to amplify target sequences from either organisms in a single amplification reaction. The co-amplification reaction, followed by separate species-specific hybridization reactions, formed the basis of an amplification/detection assay able to detect and distinguish infection with N. gonorrhoeae, C. trachomatis, or both.
The AMPLICOR.RTM. Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) Test, commercially available from Roche Diagnostic Systems, Inc. (Branchburg, N.J.), is an in vitro assay for the qualitative detection of Chlamydia trachomatis and/or Neisseria gonorrhoeae in clinical samples which is based on the assay described in the '040 patent. The AMPLICOR.RTM. Chlamydia trachomatis/Neisseria gonorrhoeae (CT/NG) Test product insert is incorporated herein by reference.
Conventional techniques of molecular biology and nucleic acid chemistry, which are within the skill of the art, are explained fully in the literature. See, for example, Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Harnes and S. J. Higgins. eds., 1984); and a series, Methods in Enzymology (Academic Press, Inc.), all of which are incorporated herein by reference. All patents, patent applications, and publications cited herein, both supra and infra, are incorporated herein by reference.